Preparation method of poplar and phellinus igniarius homogeneous polysaccharide with anti-diabetic effect

Abstract

The invention belongs to the technical field of medicine preparation, and discloses a preparation method of poplar and phellinus igniarius homogeneous polysaccharide with an anti-diabetic effect, and the preparation method comprises the following specific steps: step 1, raw material pretreatment: drying poplar and phellinus igniarius entities at 50 DEG C, and then crushing into uniform powder for subsequent extraction; and 2, extraction and purification of polysaccharide. A water extraction and alcohol precipitation method, Sevag deproteinization, dialysis, DEAE-52 cellulose, Sephadex G-100 column chromatography and other methods are adopted to separate and purify the poplar phellinus igniarius polysaccharide, a phenol sulfuric acid method and a coomassie brilliant blue method are adopted to measure the content of polysaccharide and protein, the preparation method is simple, stable and feasible, no other organic solvent is introduced, and the poplar phellinus igniarius polysaccharide is suitable for industrial production, has a very good application prospect and is suitable for industrial production. The homogeneous polysaccharide of the poplar and the phellinus igniarius has an obvious protection effect on oxidative damage of pancreatic beta cells induced by H2O2, which shows that the poplar and the phellinus igniarius have the effect of reducing blood sugar.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and specifically relates to a preparation method of homogeneous polysaccharide of Phellinus igniarius growing on poplar trees with anti-diabetic effect. Background Art

[0002] Phellinus igniarius is a precious medicinal fungus in China with a history of more than 2,000 years. It has the effects of benefiting the five internal organs, softening hardness, expelling toxins, stopping bleeding and relieving diarrhea, and is used for spleen deficiency and kidney diarrhea, sores and abscesses accumulation, hardness and mass, metrorrhagia and leukorrhagia, etc. Among different species of Phellinus igniarius, Phellinus igniarius growing on poplar trees is a medicinal fungus growing on poplar trees and also the one with the largest market share among medicinal Phellinus igniarius.

[0003] Some studies have compared the content of active ingredients and biological activities of Phellinus igniarius with other species. Experiments have proved that compared with Phellinus igniarius growing on mulberry trees, Phellinus linteus and Phellinus pini, Phellinus igniarius growing on poplar trees has higher contents of total polysaccharides, total flavonoids and total phenols, and the antioxidant activity of its extract is also higher than that of other Phellinus igniarius varieties. Therefore, its pharmacological activities have attracted great interest of researchers. The effects of anti-inflammatory, antioxidant, anti-cancer, immunomodulatory, anti-diabetic, lipid-lowering, anti-gout arthritis and anti-hyperuricemia have been well demonstrated. Therefore, it is of great significance to further improve the extraction efficiency and purification effect of polysaccharides from Phellinus igniarius growing on poplar trees and to deeply study the development and utilization of polysaccharides from Phellinus igniarius growing on poplar trees in aspects of anti-cancer, anti-tumor, antioxidant, hypoglycemic and cholesterol-lowering. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of homogeneous polysaccharide of Phellinus igniarius growing on poplar trees with anti-diabetic effect to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A preparation method of homogeneous polysaccharide of Phellinus igniarius growing on poplar trees with anti-diabetic effect, and the specific steps are as follows:

[0006] Step 1: Raw material pretreatment

[0007] Dry the fruiting body of Phellinus igniarius growing on poplar trees at 50 °C, and then crush it into a uniform powder for subsequent extraction;

[0008] Step 2: Preliminary extraction of polysaccharide

[0009] Reflux extraction: Take the crushed powder of Phellinus igniarius growing on poplar trees, add ultrapure water according to the solid-liquid ratio of 1:18, carry out reflux extraction at 90 °C for 2 hours, repeat the extraction 3 times, and combine the extraction solutions;

[0010] Water extraction and alcohol precipitation method: Concentrate the combined extraction solutions under reduced pressure to one-sixth of the original volume, then add ethanol with a mass concentration of 95% to a concentration of 80%, place it in a refrigerator at 4 °C and let it stand overnight, filter by suction, and collect the precipitate to obtain the crude polysaccharide of Phellinus igniarius growing on poplar trees;

[0011] Step 3: Deproteinization and purification of crude polysaccharide

[0012] Deproteinization by Sevag method: Dissolve the crude polysaccharide of Phellinus linteus collected in Step 2 in ultrapure water, add Sevag reagent (chloroform and n-butanol at a volume ratio of 4:1) to remove the protein in the solution, and obtain an aqueous polysaccharide solution without protein. Then, dialyze the deproteinized polysaccharide solution through a dialysis bag with a molecular weight cut-off of 3000 Da for 72 hours to remove low-molecular-weight components. Finally, freeze-dry to obtain the crude polysaccharide of Phellinus linteus that has removed protein, oligosaccharides, inorganic salts, pigments and other small-molecule impurities;

[0013] Step 4: Column chromatography purification

[0014] Preliminary separation by DEAE-52 cellulose column: Prepare an aqueous solution of the crude polysaccharide of Phellinus linteus that has removed protein, oligosaccharides, inorganic salts, pigments and other small-molecule impurities in Step 3, and separate it using a DEAE-52 cellulose column (2.5 cm × 70 cm). Gradient elution is carried out successively with ultrapure water and different concentrations of NaCl solutions (0.1, 0.2, 0.3, 0.4, 0.5, 1 mol / L) at a flow rate of 1 mL / min. Monitor the polysaccharide content in the eluate by the phenol-sulfuric acid method, collect samples according to the elution curve, collect two main polysaccharide fractions, dialyze for 48 hours, and freeze-dry after concentration;

[0015] Purification by Sephadex G-100 column: Further purify using a Sephadex G-100 column (2.5 cm × 50 cm), elute with ultrapure water at a flow rate of 0.5 mL / min, collect the eluate (10 mL / tube) according to sugar content analysis, concentrate and freeze-dry to obtain the purified product, the homogeneous polysaccharide of Phellinus linteus. The two homogeneous polysaccharides of Phellinus linteus are named SVPS-1 (5 mg) and SVPS-2 (5 mg) respectively;

[0016] Purity detection: Determine the polysaccharide and protein contents by the phenol-sulfuric acid method and Coomassie Brilliant Blue method respectively. The polysaccharide content of SVPS-1 is 97.52%, and the protein content is 1.27%; the polysaccharide content of SVPS-2 is 92.04%, and the protein content is 1.51%;

[0017] Step 5: Structure characterization

[0018] Structural characterization of two components, SVPS-1 and SVPS-2, in Phellinus linteus is carried out by ultraviolet-visible spectroscopy analysis, infrared spectroscopy analysis, scanning electron microscopy analysis, Congo red experiment method, monosaccharide composition and molecular weight determination;

[0019] Step 6: Protective effect of homogeneous polysaccharide of Phellinus linteus on pancreatic islet β cells

[0020] Through SVPS-1 and SVPS-2, H 2 O 2 To verify the efficacy of Poplar mulberry linterus homogeneous polysaccharide in the protective effect of induced INS-1 pancreatic β-cell oxidative damage, INS-1 cells were first cultured in RPMI 1640 medium containing 10% FBS, 11.1 mmol / L glucose, 100 U / ml penicillin, 0.1 mg / ml streptomycin, 0.25 μmol / ml amphotericin B, 50 μmol / L β-mercaptoethanol and 1.0 mmol / L sodium pyruvate at 37°C and 5% CO 2 The cells were cultured under the conditions of , and then the protective effect of poplar mulberry ignia polysaccharide on pancreatic β cells was observed by cell viability assay, intracellular ROS level assay, LDH and antioxidant enzyme level assay, Hoechst 33342 staining and Western blot experiment.

[0021] Preferably, the UV-visible spectroscopy analysis method in step five is performed by dissolving the two homogeneous polysaccharides SVPS-1 and SVPS-2 obtained from poplar mulberry linterus in deionized water, and measuring their UV absorption at 200-800 nm using a UV-visible spectrophotometer.

[0022] Preferably, the infrared spectroscopy analysis method in step 5 is performed by mixing 1 mg of purified poplar mulberry linterus uniform polysaccharide with KBr powder in a ratio of 1:100, grinding, mixing evenly, pressing into tablets, and scanning 4000-500 cm with KBr as the background using a Fourier transform infrared spectrometer. -1 wavelength range of the samples.

[0023] Preferably, the scanning electron microscope analysis method in step 5 is performed by fixing the SVPS-1 and SVPS-2 polysaccharide powders on a sample stage with a conductive adhesive and then performing a gold spraying treatment, and observing the microstructure of the polysaccharide under a scanning electron microscope.

[0024] Preferably, the Congo red test method described in step 5 is performed by mixing the poplar mulberry linterus uniform polysaccharide sample solution with a NaOH solution to a final concentration of 0, 0.1, 0.2, 0.3, 0.4, or 0.5 M, then adding a Congo red solution with a concentration of 80 mol / L, performing visible light absorption in the range of 200 to 800 nm, and determining the maximum absorption wavelength of the solution.

[0025] Preferably, the monosaccharide composition analysis method in step five analyzes the monosaccharide composition of the purified homogeneous polysaccharide of Phellinus vaninii by PMP derivatization and high performance liquid chromatography. Hydrolyze 5 mg of SVPS-1 and SVPS-2 with trifluoroacetic acid, and obtain the dried hydrolysis product with PMP methanol solution. The chromatographic column is HPLC-DAD, the chromatographic column is Agilent C18 (4.6 mm × 250 mm), the wavelength is 245 nm, the temperature is 30 °C, and the preparation and analysis methods of the monosaccharide standards are the same.

[0026] Preferably, the molecular weight determination method in step five determines the homogeneity and molecular weight of the purified homogeneous polysaccharide of Phellinus vaninii by using the SEC-MALLS-RI method. Dissolve 7 mg of each of SVPS-1 and SVPS-2 in 0.1 M sodium nitrate solution (containing 0.02% NaN 3 , w / w) to a final concentration of 1 mg / mL, filter through a 0.45 μm filter and then analyze. Use a combination of Ohpak SB-805HQ (300 × 8 mm) and Ohpak SB-803HQ (300 × 8 mm column) gel permeation chromatography columns for analysis. The column temperature is 45 °C, the injection volume is 100 μL, mobile phase A is 0.02% NaN 3 , 0.1 M sodium nitrate, the flow rate is 0.6 mL / min, and the elution gradient is isobaric for 75 min.

[0027] Preferably, for the cell viability assay in step six, INS-1 cells need to be seeded in a 96-well plate at a density of 2 × 10 4 cells / well. After incubation for 48 h, to study the effect of Phellinus vaninii polysaccharide on H 2 O 2 -induced cell damage, treat the cells with SVPs or 20 μM pioglitazone hydrochloride for 24 h respectively. Remove the culture medium, wash the cells with PBS, expose the cells to 100 μM of H 2 O 2 for 4 h, then add 100 μL of 5 mg / mL MTT to each well, incubate for 4 h, remove the culture medium, add 100 μL of DMSO to each well, incubate on a shaker for 10 min, and measure the absorbance of each well at 570 nm with a microplate reader.

[0028] Preferably, the intracellular ROS level determination in step six measures the production of intracellular reactive oxygen species by using the peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescein (DCFH-DA). Add DCFH-DA (10 μM) to each well and co-incubate at 37 °C for 20 min; the determination of LDH and antioxidant enzyme levels detects the intracellular LDH, SOD, CAT, and GSH levels according to the kit instructions.

[0029] Preferably, the Hoechst 33342 staining method described in step 6 is performed by observing the nuclear morphology of I NS-1 cells and using the DNA-specific dye Hoechst 33342 staining method to obtain a 6-well plate at a density of 2.5×10 5 INS-1 cells were treated with or without SVPs or PH for 24 h and exposed to 100 μM PH. 2 O 2 The cells were incubated for 4 h at 37 °C in the dark, stained with Hoechst 33342 for 10 min, and the nuclear morphology was observed under a 20× fluorescence microscope to determine the degree of nuclear condensation. In the Western blotting experiment, the total protein of each group of cells was extracted, the concentration was determined by the BCA method, and the SDS-PAGE gel electrophoresis method was used according to a sample load of 40 μg. The membrane was wet-transferred to a PVDF membrane and blocked for 2 h. The membrane was washed with TBST 3-5 times for 10 min each time, incubated with primary antibody at 4 °C, shaken on a low-speed shaker overnight, washed with TBST, added with goat anti-mouse or goat anti-rabbit secondary antibody, incubated at room temperature for 1 h, washed with TBST 3 times, exposed by ECL chemiluminescence, and analyzed using the image analysis software provided with the Fusion FX Spectra system.

[0030] The beneficial effects of the present invention are as follows:

[0031] The polysaccharide from Phellinus igniarius was isolated and purified by water extraction and alcohol precipitation, Sevag deproteinization, dialysis, DEAE-52 cellulose and Sephadex G-100 column chromatography, and the polysaccharide and protein contents were determined by phenol-sulfuric acid method and Coomassie brilliant blue method, respectively. The preparation method is simple, stable and feasible, does not introduce other organic solvents, is suitable for industrial production, and has a good application prospect. The single polysaccharide from Phellinus igniarius has a good effect on H 2 O 2 It has a significant protective effect on the oxidative damage induced by pancreatic beta cells, indicating that poplar mulberry yellow has the effect of lowering blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a DEAE-52 column chromatography elution curve of the crude polysaccharide of poplar mulberry linterus of the present invention;

[0033] Figure 2 The elution curves of SVPS-1 (a) and SVPS-2 (b) of the present invention on Sephadex column chromatography;

[0034] Figure 3 The UV spectra of SVPS-1 and SVPS-2 of the present invention are shown in FIG.

[0035] Figure 4 The infrared spectra of SVPS-1 and SVPS-2 of the present invention are shown in FIG.

[0036] Figure 5 These are the scanning electron microscope images of SVPS-1 and SVPS-2 of the present invention;

[0037] Figure 6 These are the Congo red curve spectra of SVPS-1 and SVPS-2 of the present invention;

[0038] Figure 7 These are the monosaccharide composition analysis spectra of SVPS-1 and SVPS-2 of the present invention;

[0039] Figure 8 These are the molecular weight determination spectra of SVPS-1(a) and SVPS-2(b) of the present invention;

[0040] Figure 9 These are the cell viability determination images of the present invention;

[0041] Figure 10 These are the images of the effect of SVPS-1 of the present invention on the production of intracellular ROS in INS-1 cells;

[0042] Figure 11 These are the images of the effect of SVPS-1 of the present invention on the contents of LDH, MDA, SOD, CAT and GSH in H 2 O 2 induced INS-1 cells;

[0043] Figure 12 These are the schematic diagrams of the effect of the polysaccharide extract of Phellinus igniarius on apoptosis of H 2 O 2 induced INS-1 cells of the present invention;

[0044] Figure 13 These are the schematic diagrams of the effect of SVPS-1 of the present invention on the expression of apoptosis proteins and insulin secretion pathway proteins in pancreatic islet β cells. Detailed implementation manners

[0045] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] As Figures 1 to 13 shown, the embodiments of the present invention provide a preparation method of a Phellinus igniarius homogeneous polysaccharide with antidiabetic effects, and the specific steps are as follows:

[0047] Step 1: Raw material pretreatment

[0048] Dry the Phellinus linteus fruiting body at 50 °C and then crush it into a uniform powder for subsequent extraction;

[0049] Step 2: Preliminary extraction of polysaccharides

[0050] Reflux extraction: Take the crushed Phellinus linteus powder, add ultrapure water according to the solid-liquid ratio of 1:18, and perform reflux extraction at 90 °C for 2 hours. Repeat the extraction 3 times and combine the extracts;

[0051] Water extraction and alcohol precipitation method: Concentrate the combined extracts under reduced pressure to one-sixth of the original volume, then add 4 times the volume of ethanol with a mass concentration of 95% until the concentration reaches 80%, place it in a refrigerator at 4 °C and let it stand overnight, filter by suction, and collect the precipitate to obtain the crude polysaccharide of Phellinus linteus;

[0052] Step 3: Deproteinization and purification of crude polysaccharides

[0053] Deproteinization by Sevag method: Dissolve the crude polysaccharide of Phellinus linteus collected in Step 2 in ultrapure water, add Sevag reagent (chloroform and n-butanol with a volume ratio of 4:1) to remove the protein in the solution, obtain an aqueous polysaccharide solution without protein, then dialyze the deproteinized polysaccharide solution through a dialysis bag with a molecular weight cut-off of 3000 Da for 72 hours to remove low molecular weight components, and finally obtain the crude polysaccharide of Phellinus linteus that has removed protein, oligosaccharides, inorganic salts, pigments and other small molecule impurities after freeze-drying;

[0054] Step 4: Column chromatography purification

[0055] Preliminary separation by DEAE-52 cellulose column: Prepare an aqueous solution of the crude polysaccharide of Phellinus linteus that has removed protein, oligosaccharides, inorganic salts, pigments and other small molecule impurities in Step 3, separate it using a DEAE-52 cellulose column (2.5 cm × 70 cm), and perform gradient elution successively with ultrapure water and different concentrations of NaCl solutions (0.1, 0.2, 0.3, 0.4, 0.5, 1 mol / L) at a flow rate of 1 mL / min. Monitor the polysaccharide content in the eluate by the phenol-sulfuric acid method, collect samples according to the elution profile, collect two main polysaccharide fractions, dialyze for 48 hours, concentrate and freeze-dry;

[0056] Fine purification by Sephadex G-100 column: Further purify using a Sephadex G-100 column (2.5 cm × 50 cm), elute with ultrapure water at a flow rate of 0.5 mL / min, collect the eluate (10 mL / tube) according to the sugar content analysis, concentrate and freeze-dry to obtain the purified product, the homogeneous polysaccharide of Phellinus linteus. The two homogeneous polysaccharides of Phellinus linteus are named SVPS-1 (5 mg) and SVPS-2 (5 mg) respectively;

[0057] Purity detection: The phenol-sulfuric acid method and Coomassie Brilliant Blue method were used to determine the polysaccharide and protein contents respectively. The polysaccharide content of SVPS-1 was 97.52%, and the protein content was 1.27%; the polysaccharide content of SVPS-2 was 92.04%, and the protein content was 1.51%.

[0058] Step Five: Structural characterization

[0059] The structures of two components, SVPS-1 and SVPS-2, in Phellinus tremulae were characterized by ultraviolet-visible spectroscopy analysis, infrared spectroscopy analysis, scanning electron microscopy analysis, Congo red assay, monosaccharide composition and molecular weight determination.

[0060] Step Six: Efficacy verification

[0061] The efficacy of the homogeneous polysaccharides from Phellinus tremulae was verified by the protective effects of SVPS-1 and SVPS-2 on H 2 O 2 induced oxidative damage of INS-1 pancreatic islet β cells. First, INS-1 cells were cultured in RPMI 1640 medium containing 10% FBS, 11.1 mmol / L glucose, 100 U / ml penicillin, 0.1 mg / ml streptomycin, 0.25 μmol / ml amphotericin B, 50 μmol / L β-mercaptoethanol and 1.0 mmol / L sodium pyruvate at 37°C and 5% CO 2 Then, it was verified by cell viability assay, intracellular ROS level assay, LDH and antioxidant enzyme level assay, Hoechst 33342 staining method and Western blot experiment.

[0062] The polysaccharides from Phellinus tremulae were isolated and purified by methods such as water extraction and alcohol precipitation, Sevag deproteinization, dialysis, DEAE-52 cellulose and Sephadex G-100 column chromatography. The DEAE-52 cellulose column chromatography was eluted step by step with ultrapure water and NaCl solution. The polysaccharide content of each group was determined by the phenol-sulfuric acid method. Two main fractions were collected, dialyzed with water, concentrated and freeze-dried. Then, the two fractions were loaded onto a Sephadex G-100 column for purification, eluted with ultrapure water, and the eluate was collected according to the polysaccharide content analysis, concentrated and freeze-dried. The purified products obtained were named SVPS-1 and SVPS-2 respectively, and the polysaccharide and protein contents were determined by the phenol-sulfuric acid method and Coomassie Brilliant Blue method respectively.

[0063] Among them, in step five, the ultraviolet-visible spectroscopy analysis method was to dissolve the two obtained homogeneous polysaccharides, SVPS-1 and SVPS-2, from Phellinus tremulae in deionized water, and use an ultraviolet-visible spectrophotometer to measure their ultraviolet absorption at 200 - 800 nm.

[0064] It can be seen from the UV spectra of SVPS-1 and SVPS-2 that SVPS-1 has a weak absorption peak at 280 nm, indicating that there may be trace amounts of protein in it. There is no absorption peak in the range of 260 - 280 nm for SVPS-2, indicating that SVPS-2 does not contain nucleic acid and protein. The results of UV spectral analysis show that the purity of the two components is relatively high.

[0065] Among them, in the infrared spectral analysis method of step five, 1 mg of purified Phellinus linteus homogeneous polysaccharide is mixed with KBr powder in a ratio of 1:100, ground and mixed evenly, pressed into a tablet, and the sample in the wavelength range of 4000 - 500 cm -1 is scanned using a Fourier transform infrared spectrometer with KBr as the background.

[0066] It can be seen from the infrared spectra of SVPS-1 and SVPS-2 that the absorption peaks at 3425 and 3412 cm -1 are the stretching vibrations of the O-H bond; the absorption peaks at 2928 and 2924 cm -1 are the stretching vibration peaks of the C-H bond; the absorption peak near 1635 cm -1 is caused by the stretching vibration of the C=O bond, and the absorption peaks near 1421 and 1415 cm -1 are the angular vibration peaks of C-H; the absorption peaks at 1000 - 1200 cm -1 are the characteristic absorption peaks where the pyranose ring overlaps with the C-O-C vibration and the C-O-H stretching vibration; there are absorption peaks at 874 and 871 cm -1 , indicating that SVPS-1 and SVPS-2 belong to the β structure; the peaks near 820 cm -1 indicate the presence of the α configuration.

[0067] Among them, in the scanning electron microscope analysis method of step five, the polysaccharide powders of SVPS-1 and SVPS-2 are fixed on the sample stage with conductive glue and then sputter-coated with gold, and the microscopic structure of the polysaccharide is observed under the scanning electron microscope.

[0068] In the scanning electron microscope images of SVPS-1 and SVPS-2, the magnification at point A is 500 times; the magnification at point B is 2000 times; the magnification at point C is 5000 times. It can be seen that when the magnification is 500 times, the microscopic morphologies of the two polysaccharides both show irregular blocky structures. After magnifying to 2000 times and 5000 times, the surfaces of the polysaccharides are rough and have a small number of small holes.

[0069] Among them, in step five, the Congo red test method determines the maximum absorption wavelength of the solution by mixing the homogeneous polysaccharide sample solution of Phellinus linteus with NaOH solution to a final concentration of 0, 0.1, 0.2, 0.3, 0.4, 0.5 M, then adding Congo red solution with a concentration of 80 mol / L, and performing visible light absorption in the range of 200 to 800 nm.

[0070] Congo red is an acidic dye that can complex with substances having a triple helix structure. It can be seen from the Congo red curve spectra of SVPS-1 and SVPS-2 that when the NaOH concentration gradually increases, the maximum absorption wavelength also increases, indicating that SVPS-1 and SVPS-2 have a triple helix structure.

[0071] Among them, the monosaccharide composition analysis method in step five analyzes the monosaccharide composition of the purified homogeneous polysaccharide of Phellinus linteus by PMP derivatization and high performance liquid chromatography. Hydrolyze 5 mg of SVPS-1 and SVPS-2 with trifluoroacetic acid, and obtain the dried hydrolysis product with PMP methanol solution. The chromatographic column is HPLC-DAD, the chromatographic column is Agilent C18 (4.6 mm × 250 mm), the wavelength is 245 nm, the temperature is 30 °C, and the preparation and analysis methods of the monosaccharide standards are the same.

[0072] In the mixed standard of the monosaccharide composition analysis spectra of SVPS-1 and SVPS-2, 1 is mannose, 2 is ribose, 3 is rhamnose, 4 is glucuronic acid, 5 is sorbose, 6 is galacturonic acid, 7 is glucose, 8 is galactose, 9 is arabinose, and 10 is xylose;

[0073]

[0074] Through the upper surface and the attached instructions Figure 7 It can be seen that SVPS-1 is composed of mannose, rhamnose, galacturonic acid, glucose, arabinose and xylose, and the molar ratio of each monosaccharide is 7.9:6.25:2.83:1.63:1.55:1; SVPS-2 has the same monosaccharide composition, and the molar ratio of monosaccharides is 7.97:12.58:7.96:3.87:2.69:1.38:1; The results show that both SVPS-1 and SVPS-2 contain galacturonic acid, proving that the purified homogeneous polysaccharide of Phellinus linteus is an acidic polysaccharide.

[0075] Among them, the molecular weight determination method in step five measures the homogeneity and molecular weight of the purified Phellinus vaninii polysaccharide by using the SEC-MALLS-RI method. 7 mg each of SVPS-1 and SVPS-2 were dissolved in 0.1 M sodium nitrate solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, filtered through a 0.45 μm filter and then analyzed. A combination of Ohpak SB-805HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm column) gel permeation chromatography columns was used for analysis. The column temperature was 45 °C, the injection volume was 100 μL, the mobile phase A was 0.02% NaN3, 0.1 M sodium nitrate, the flow rate was 0.6 mL / min, and the elution gradient was isobaric for 75 min.

[0076]

[0077] The molecular weights of SVPS-1 and SVPS-2 polysaccharides were determined using a gel chromatography-differential-multi-angle laser scattering system. From the above table and the attached Figure 8 It can be seen that the molecular weights of SVPS-1 and SVPS-2 are 32.069 and 144.625 kDa respectively, and the Mw / Mn values are 1.120 and 1.414 respectively, showing a single-peak symmetry, indicating high component purity and a relatively narrow molecular weight distribution.

[0078] Among them, for the cell viability determination in step six, INS-1 cells were seeded in 96-well plates at a density of 2×10 4 cells / well and incubated for 48 h. To study the effect of Phellinus vaninii polysaccharide on H 2 O 2 -induced cell damage, the cells were treated with SVPs or 20 μM pioglitazone hydrochloride for 24 h respectively. The medium was removed, the cells were washed with PBS, and the cells were exposed to 100 μM H 2 O 2 for 4 h. Then, 100 μL of 5 mg / mL MTT was added to each well and incubated for 4 h. The medium was removed, 100 μL of DMSO was added to each well, and incubated on a shaker for 10 min. The absorbance of each well was measured with a microplate reader at 570 nm.

[0079] In the cell viability determination graph, a and b are the effects of SVPS-1 and SVPS-2 on the survival rate of INS-1 cells respectively, c is the effect of different concentrations of H 2 O 2 on the survival rate of INS-1 cells, and d and e are the effects of SVPS-1 and SVPS-2 on H 2 O 2Effect on the viability of induced INS-1 cells. Compared with the untreated group, the cell viability of the SVPS-1 group was better than that of the SVPS-2 group (p < 0.05); when the concentrations of the two polysaccharide components were 6.25 - 50 μg / mL, the cell viability was above 90%, indicating that the two polysaccharide components had no obvious cytotoxicity to INS-1 cells within a certain concentration range. Therefore, the treatment concentrations of SVPS-1 and SVPS-2 were selected as 6.25 - 50 μg / mL in the subsequent experiments; to establish an INS-1 cell model of oxidative stress injury, INS-1 cells were treated with different concentrations of (12.5 - 800 μM) H 2 O 2 . Compared with the blank group c, the cell viability of the 100 μM treatment group was 50.21%. Therefore, 100 μM of H 2 O 2 was used in the subsequent experiments to establish a cell injury model. The experimental results of the effect of SVPS-1 and SVPS-2 on the viability of H 2 O 2 -induced INS-1 cells showed that compared with the normal group, the cell viability of the H 2 O 2 treatment group was significantly reduced (p < 0.001). After pretreatment with 6.25 - 50 μg / mL SVPS-1 and SVPS-2, the cell viabilities were 71.18% / 57.02%, 73.85% / 62.67%, 83.19% / 76.39, and 90.52% and 83.74% respectively, indicating that SVPS-1 had a better protective effect on cell damage.

[0080] Among them, the intracellular ROS level in step six was measured by using the peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescein (DCFH-DA) to measure the production of intracellular reactive oxygen species. DCFH-DA (10 μM) was added to each well and co-incubated at 37 °C for 20 min; the levels of LDH and antioxidant enzymes were measured by following the kit instructions to detect the levels of intracellular LDH, SOD, CAT, and GSH.

[0081] In the attached instructions Figures 10 to 11 , * indicates a significant difference compared with the model group (p < 0.05), ** indicates a relatively significant difference compared with the model group (p < 0.01), *** indicates a highly significant difference compared with the model group (p < 0.001), # indicates a significant difference compared with the blank group (p < 0.05), ## indicates a relatively significant difference compared with the blank group (p < 0.01), indicates a highly significant difference compared with the blank group (p < 0.001);

[0082] The measurement of ROS production was performed by using the DCFH-DA method to detect the effect of SVPS-1 on the intracellular ROS production of INS-1 cells, as described in the attached instructionsFigure 10 As shown, compared with untreated cells, H 2 O 2 treatment can significantly increase the production of ROS in INS-1 cells, and the ROS level is 2.75 times that of the control group. After pre-treating the cells with SVPS-1 and pioglitazone hydrochloride at the specified concentrations, the production of ROS decreased significantly in a dose-dependent manner. At a concentration of 50 μg / mL, SVPS-1 pretreatment reduced the ROS level to 1.3 times that of the control group; The effects of SVPS-1 on oxidative stress-related biochemical indicators are as shown in the attached instructions Figure 11 As shown, H 2 O 2 treatment led to a significant increase in the production of LDH and MDA in INS-1 cells. Pretreatment with 6.25, 12.5, 25, and 50 μg / mL of SVPS-1 could significantly reduce the H 2 O 2 -induced LDH and MDA contents. The results of the determination of the activities of antioxidant enzymes SOD, CAT, and GSH showed that compared with the control group, the SOD activity in the hydrogen peroxide treatment group decreased significantly. After pretreatment with SVPS-1, the SOD level in the cells increased significantly. Compared with the NC group, the CAT activity in the SVPS-1 pretreatment group increased significantly. At a concentration of 50 μg / mL, the CAT level decreased to 70% of the control group. As can be seen from the figure, H 2 O 2 treatment could significantly reduce the GSH level, while after pretreatment with SVPS-1, the GSH content increased significantly. When the concentration reached 50 μg / mL, the GSH level increased significantly (75% of the control group). The results also showed that 20 μM of pioglitazone hydrochloride could effectively improve the antioxidant enzyme activity of H 2 O 2 -induced INS-1 cells. The above results indicate that SVPS-1 has an obvious protective effect on INS-1 cells by increasing the reducing activity of antioxidant enzymes and reducing the production of LDH and MDA.

[0083] Among them, the Hoechst 33342 staining method in step six observes the nuclear morphology of INS-1 cells. Using the DNA-specific dye Hoechst 33342 staining method, INS-1 cells with a density of 2.5×10 5 cells / well were taken in a 6-well plate, and were treated with or without SVPs or PH for 24 h, and exposed to 100 μM of H 2 O 2The cells were incubated for 4 h at 37 °C in the dark, stained with Hoechst33342 for 10 min, and the nuclear morphology was observed under a 20× fluorescence microscope to determine the degree of nuclear condensation. In the Western blotting experiment, the total protein of each group of cells was extracted, the concentration was determined by the BCA method, and the SDS-PAGE gel electrophoresis method was used according to a loading amount of 40 μg. The membrane was wet-transferred to a PVDF membrane and blocked for 2 h. The membrane was washed with TBST 3-5 times for 10 min each time, incubated with the primary antibody at 4 °C, shaken on a low-speed shaker overnight, washed with TBST, added with goat anti-mouse or goat anti-rabbit secondary antibody, incubated at room temperature for 1 h, washed with TBST 3 times, exposed with ECL chemiluminescence, and analyzed with the image analysis software provided with the Fusion FX Spectra system.

[0084] As the instruction manual Figure 12 As shown in the Hoechst 33342 staining, living cells with intact nuclei and intact nuclei can be observed in the control group, while in the H 2 O 2 In the treatment group, more fluorescent spots could be observed, suggesting nuclear fragmentation and chromatin condensation, while pretreatment with different concentrations of SVPS-1 and pioglitazone hydrochloride reduced cell apoptosis, with fewer blue spots visible under the microscope. The results showed that SVPS-1 had a protective effect on oxidative stress-induced apoptosis of INS-1 cells;

[0085] Western blotting results are as shown in the instruction manual. Figure 13 As shown in the figure, compared with the blank group, the expression of Bax protein in pancreatic β cells in the model group was significantly increased, and the expression level of Bc l-2 protein was decreased, indicating that the cell apoptosis was significantly higher than that in the blank group. Compared with the model group, the expression of Bax in cells pretreated with 25 and 50 μg / mL SVPS-1 was significantly decreased, and the expression of Bc l-2 protein was significantly increased, among which the protein expression level of the 50 μg / mL treatment group was equivalent to that of the positive control drug treatment group. Compared with the blank group, the expression of caspase-9 and caspase-3 proteins in pancreatic β cells in the model group was significantly increased, and the expression of caspase-9 and caspase-3 proteins in cells pretreated with SVPS-1 was significantly decreased, indicating that SVPS-1 can regulate cell apoptosis-related proteins to play its cytoprotective role. Compared with the blank group, the expression levels of PDX-1 and GLUT4 proteins in pancreatic β cells in the model group were significantly decreased; compared with the model group, the expression levels of PDX-1 and GLUT4 proteins in each SVPS-1 treatment group were increased, indicating that SVPS-1 can regulate the proteins related to the insulin secretion pathway to play its cytoprotective role.

[0086] It should be noted that in this text, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus.

[0087] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing poplar phellinus linteus homogeneous polysaccharide with anti-diabetic effect, characterized in that: The specific steps are as follows: Step 1: Raw material pretreatment The poplar phellinus linterus entities were dried at 50°C and then crushed into uniform powder for subsequent extraction; Step 2: Preliminary extraction of polysaccharides Reflux extraction: Take the crushed poplar mulberry powder, add ultrapure water at a solid-liquid ratio of 1:18, and reflux extract at 90°C for 2 hours. Repeat the extraction 3 times and combine the extracts; Water extraction and alcohol precipitation method: The combined extract was concentrated to one sixth of the original volume under reduced pressure, and then 95% ethanol was added to a concentration of 80%, and the mixture was placed in a refrigerator at 4°C and allowed to stand overnight. The precipitate was collected and filtered to obtain the crude polysaccharide of Phellinus igniarius from poplar trees. Step 3: Deproteinization and purification of crude polysaccharides Deproteinization by Sevag method: dissolving the crude polysaccharide of Phellinus igniarius collected in step 2 in ultrapure water, adding Sevag reagent (chloroform and n-butanol 4:1 volume ratio) to remove the protein in the solution to obtain a polysaccharide aqueous solution after protein removal, and then dialyzing the deproteinized polysaccharide solution through a dialysis bag with a molecular weight cutoff of 3000Da for 72 hours to remove low molecular weight components, and finally freeze-drying to obtain the crude polysaccharide of Phellinus igniarius from which small molecular impurities such as protein, oligosaccharides, inorganic salts, and pigments have been removed; Step 4: Column chromatography purification Preliminary separation using a DEAE-52 cellulose column: The crude polysaccharide from poplar mulberry linterus, from which small molecular impurities such as proteins, oligosaccharides, inorganic salts, and pigments have been removed in step 3, is prepared into an aqueous solution, separated using a DEAE-52 cellulose column (2.5 cm × 70 cm), and gradient eluted with ultrapure water and different concentrations of NaCl solution (0.1, 0.2, 0.3, 0.4, 0.5, 1 mol / L) in sequence, at a flow rate of 1 mL / min. The polysaccharide content of the eluate is monitored using the phenol-sulfuric acid method, and samples are collected according to the elution curve. Two main polysaccharide fractions are collected, dialyzed for 48 hours, concentrated, and then freeze-dried; Sephadex G-100 column purification: Sephadex G-100 column (2.5 cm × 50 cm) was used for further purification, eluted with ultrapure water at a flow rate of 0.5 mL / min, and the eluate (10 mL / tube) was collected according to the polysaccharide content analysis, concentrated and freeze-dried to obtain the purified product poplar phellinus linteus homogeneous polysaccharide. The two poplar phellinus linteus homogeneous polysaccharides were named SVPS-1 (5 mg) and SVPS-2 (5 mg), respectively; Purity detection: The polysaccharide and protein contents were determined by phenol-sulfuric acid method and Coomassie brilliant blue method, respectively. The polysaccharide content of SVPS-1 was 97.52%, and the protein content was 1.27%; the polysaccharide content of SVPS-2 was 92.04%, and the protein content was 1.51%; Step 5: Structural Characterization The structures of SVPS-1 and SVPS-2 in Poplar Coriolus linteus were characterized by UV-visible spectroscopy, infrared spectroscopy, scanning electron microscopy, Congo red test, monosaccharide composition and molecular weight determination. Step 6: Protective effect of poplar mulberry linterus homogeneous polysaccharide on pancreatic β cells The efficacy of poplar mulberry igniarius homogeneous polysaccharide was verified by the protective effect of SVPS-1 and SVPS-2 on H2O2-induced oxidative damage of INS-1 pancreatic β cells. First, INS-1 cells were cultured in RPMI 1640 culture medium, which contained 10% FBS, 11.1 mmol / L glucose, 100 U / ml penicillin, 0.1 mg / ml streptomycin and 0.25 μmol / ml amphotericin B, 50 μmol / L β-mercaptoethanol and 1.0 mmol / L sodium pyruvate at 37°C and 5% CO2. Then, the protective effect of poplar mulberry igniarius polysaccharide on pancreatic β cells was observed by cell viability assay, intracellular ROS level assay, LDH and antioxidant enzyme level assay, Hoechst 33342 staining and Western blot experiment.

2. The method for preparing the poplar phellinus linteus homogeneous polysaccharide with anti-diabetic effect according to claim 1, characterized in that: The UV-visible spectroscopy analysis method described in step 5 is performed by dissolving the two homogeneous polysaccharides SVPS-1 and SVPS-2 obtained from poplar mulberry linterus in deionized water, and measuring the UV absorption thereof at 200-800 nm using a UV-visible spectrophotometer.

3. The method for preparing the poplar phellinus linteus homogeneous polysaccharide with anti-diabetic effect according to claim 1, characterized in that: The infrared spectroscopy analysis method in step 5 is as follows: 1 mg of purified poplar mulberry linterus homogeneous polysaccharide is mixed with KBr powder in a ratio of 1:100, ground, mixed evenly, pressed into tablets, and scanned at 4000-500 cm with KBr as the background using a Fourier transform infrared spectrometer. -1 wavelength range of the samples.

4. The method for preparing the poplar phellinus linteus homogeneous polysaccharide with anti-diabetic effect according to claim 1, characterized in that: The scanning electron microscope analysis method described in step 5 is to fix the SVPS-1 and SVPS-2 polysaccharide powders on the sample stage with conductive glue and then perform gold spraying treatment, and observe the microstructure of the polysaccharide under a scanning electron microscope.

5. The method for preparing the poplar phellinus linteus uniform polysaccharide with anti-diabetic effect according to claim 1, characterized in that: The Congo red test method described in step 5 is to mix the poplar mulberry linterus uniform polysaccharide sample solution with the NaOH solution to a final concentration of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 M, then add a Congo red solution with a concentration of 80 mol / L, absorb visible light in the range of 200 to 800 nm, and determine the maximum absorption wavelength of the solution.

6. The method for preparing the poplar phellinus linteus uniform polysaccharide with anti-diabetic effect according to claim 1, characterized in that: The monosaccharide composition analysis method described in step five is to purify the monosaccharide composition of poplar mulberry linterus uniform polysaccharide through PMP derivatization and high performance liquid chromatography analysis, hydrolyze 5 mg of SVPS-1 and SVPS-2 with trifluoroacetic acid, and use PMP methanol solution to obtain a dry hydrolyzate. The chromatographic column is HPLC-DAD, the chromatographic column is Agilent C18 (4.6mm×250mm), the wavelength is 245nm, the temperature is 30°C, and the preparation and analysis methods of monosaccharide standards are the same.

7. The method for preparing the poplar phellinus linteus uniform polysaccharide with anti-diabetic effect according to claim 1, characterized in that: The molecular weight determination method described in step five is to determine the uniformity and molecular weight of the purified poplar mulberry linterus uniform polysaccharide by using the SEC-MALLS-RI method. 7 mg each of SVPS-1 and SVPS-2 is dissolved in 0.1 M sodium nitrate solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and analyzed after filtering through a 0.45 μm filter. The analysis is performed using a combination of Ohpak SB-805HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm column) gel exclusion chromatography columns. The column temperature is 45°C, the injection volume is 100 μL, the mobile phase A is 0.02% NaN3, 0.1 M sodium nitrate, the flow rate is 0.6 mL / min, and the elution gradient isobaric for 75 min.

8. The method for preparing the poplar phellinus linteus uniform polysaccharide with anti-diabetic effect according to claim 1, characterized in that: The cell viability assay described in step 6 requires that INS-1 cells be plated at 2 × 10 4 The cells were inoculated at a density of 100 μg / well in a 96-well plate and incubated for 48 h. In order to study the effect of poplar mulberry ignia polysaccharide on H2O2-induced cell damage, the cells were treated with SVPs or 20 μM pioglitazone hydrochloride for 24 h, the culture medium was removed, the cells were washed with PBS, the cells were exposed to 100 μM H2O2 for 4 h, and then 100 μL of 5 mg / mL MTT was added to each well and incubated for 4 h. The culture medium was removed, 100 μL of DMSO was added to each well, and the plates were incubated on a shaker for 10 min. The absorbance of each well was measured at 570 nm using a microplate reader.

9. The method for preparing the poplar phellinus linteus uniform polysaccharide with anti-diabetic effect according to claim 1, characterized in that: The intracellular ROS level described in step six was determined by using the peroxide-sensitive fluorescent probe 2′, 7′ dichlorofluorescein (DCFH-DA) to measure the production of intracellular reactive oxygen species. DCFH-DA (10 μM) was added to each well and incubated at 37°C for 20 minutes. The LDH and antioxidant enzyme levels were determined by detecting the intracellular LDH, SOD, CAT, and GSH levels according to the kit instructions.

10. The method for preparing the poplar phellinus linteus uniform polysaccharide with anti-diabetic effect according to claim 1, characterized in that: The Hoechst 33342 staining method described in step 6 was performed by observing the nuclear morphology of INS-1 cells and using the DNA-specific dye Hoechst 33342 staining method. The density was 2.5×10 5 INS-1 cells per well were treated with or without SVPS or pioglitazone hydrochloride for 24 h, and exposed to 100 μM H2O2 for 4 h at 37°C in the dark. They were stained with Hoechst33342 for 10 min, and the nuclear morphology was observed under a 20× fluorescence microscope to determine the degree of nuclear condensation. In Western blot experiments, total protein from each group of cells was extracted, and the concentration was determined by the BCA method. According to a loading amount of 40 μg, the SDS-PAGE gel electrophoresis method was used, and the membrane was wet-transferred to PVDF membrane, blocked for 2 h, washed with TBST 3-5 times, each time for 10 min, incubated with primary antibody at 4°C, shaken on a low-speed shaker overnight, washed with TBST, added with goat anti-mouse or goat anti-rabbit secondary antibody, incubated at room temperature for 1 h, washed with TBST 3 times, exposed by ECL chemiluminescence, and analyzed using the image analysis software provided with the Fusion FX Spectra system.

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